人脐带间充质干细胞对HaCaT细胞增殖与迁移的影响

Effects of human umbilical cord mesenchymal stem cells on the proliferation and migration of HaCaT cells

目的探讨人脐带间充质干细胞(hucMSCs)对人表皮角质形成细胞增殖与迁移的影响及其可能机制。方法采用Ⅱ型胶原酶消化法从新生儿脐带中分离制备hucMSCs,用流式细胞术鉴定P3代hucMSCs表面标记抗原,用油红O染色、茜素红S染色和Masson染色进行间充质干细胞三向分化能力鉴定;以人表皮角质细胞系HaCaT为研究模型,在transwell上室接种P3代hucMSCs进行共培养,显微镜观察24、48和72 h时HaCaT的细胞形态,CCK8法检测细胞增殖状况,划痕法检测细胞12 h和24 h迁移率,qRT-PCR法测定HaCaT细胞迁移、增殖相关生长因子KGF-2、FGFR-2、TGF-β1,以及细胞外基质蛋白Fibronectin、MMP-1和CollagenⅠmRNA表达水平。结果hucMSCs呈成纤维细胞样贴壁生长,其表面间充质干细胞标志性抗原CD105、CD90、CD73阳性表达率分别为98.35%、99.97%和99.94%,不表达CD45、CD34、CD14和CD19,在诱导培养16d后出现明显的成脂、成骨和成软骨细胞特性。hucMSCs共培养24、48和72 h,HaCaT细胞存活率较对照组分别显著增加了(20.42±3.90)%(P=0.002)、(36.30±8.08)%(P=0.001)和(27.31±10.04)%(P=0.012),与形态学观察结果一致。划痕后继续培养12h,对照组、共培养组HaCaT的空白区域面积分别为(36354.19±1705.32)μm^2、(29497.63±1286.49)μm^2(P=0.045),培养24 h,其空白区域面积分别减少为(13086.65±1695.85)μm^2、(2895.69±224.32)μm^2(P=0.014)。qRT-PCR结果显示,共培养处理24h后huc MSCs组KGF-2、TGF-β1基因表达分别上调为对照组的1.24倍和1.92倍;共培养48 h后KGF-2、TGF-β1和FGFR-2分别上调2.01倍、2.26倍和1.34倍,细胞外基质蛋白Fibronectin转录水平显著上调为对照组的1.59倍,MMP-1降低至对照组的0.52倍。结论hucMSCs能够显著促进HaCaT细胞的增殖与迁移,这可能与hucMSCs调节HaCaT增殖、迁移相关生长因子及细胞外基质蛋白的基因表达水平有关,提示hucMSCs是有潜力的表皮创伤愈合种子细胞。

Objective To investigate the effects of human umbilical cord mesenchymal stem cells(hucMSCs)on the proliferation and migration of human epidermal keratinocytes and its potential mechanisms.Methods HucMSCs were isolated from neonatal umbilical cord using type II collagenase digestion method.Flow cytometry was applied to identify the characteristic antigenic markers of P3-generation hucMSCs.Oil red O staining,alizarin red S staining and Masson staining were used to identify the differentiation abilities of hucMSCs.Then,HaCaT keratinocytes and hucMSCs were co-cultured using a transwell membrane.After 24,48 and 72 h,the cell viability of HaCaT was measured by CCK8 assay and cell morphology was photographed.Moreover,scratch assay was applied to determine the migration of HaCaT cells co-cultured with huc MSCs for 12 h and 24 h.The mRNA expression of growth factors associated with cell migration,proliferation and extracellular matrix proteins were further characterized by qRT-PCR.Results P3-generation hucMSCs showed fibroblast-like adherent growth and expression of CD105,CD90 and CD73,while lack of CD45,CD34,CD14 and CD19.After 16 d of induction culture,lipogenic,osteogenic and chondrogenic characteristics were also observed.Consistent with the morphologic observations,the viability of HaCaT cells co-cultured with hucMSCs was significantly increased for(20.42±3.90)%(P=0.002),(36.30±8.08)%(P=0.001)and(27.31±10.04)%(P=0.012)for 24,48 and 72 h,respectively.Wound scrath assay revealed that huc MSCs significantly promoted the migration of HaCaT after 12 h and 24 h incubation(P<0.05).Furthermore,the mRNA expression of KGF-2 and TGF-β1 in HaCaT were up-regulated by 1.24 and 1.92 folds after 24 h co-culture with hucMSCs,KGF-2,TGF-β1,FGFR-2 and Fibronectin mRNA expression were up-regulated by 2.01,2.26,1.34 and 1.59 folds after 48 h co-culture,while MMP-1 was down-regulated by 0.52 folds.Conclusion HucMSCs can significantly promote the proliferation and migration of HaCaT cells,which may be related to regulation of keratinocytes proliferation,migration-related growth factors and extracellular matrix proteins m RNA expression in HaCaT.These results suggest that hucMSCs be potential seed cells for epidermal wound healing.